Comparison and optimization of extraction protocol for intracellular phosphorus and its polyphosphate in enhanced biological phosphorus removal (EBPR) sludge

Abstract

Intracellular polyphosphate (poly-P) plays important roles in Enhanced biological phosphorus removal (EBPR) process, but an effective and reliable protocol for extracting intracellular P and its poly-P in EBPR sludge without hydrolysis of poly-P has not been setup yet. In the study, it was revealed that the severe hydrolysis of intracellular poly-P occurred during the different extraction processes, such as acid (i.e., HClO4, H2SO4 and HCl), basic (i.e., NaOH and KOH) and freezing-grind (under different solid-liquid ratios), but it did not occur during ultrasonic extraction process. The optimal extraction process of the ultrasonic protocol was 10 w/mL of ultrasonic power density and 15 min of ultrasonic time, when the extraction efficiency of intracellular P was 88.24 ± 1.56%. In addition, the extraction efficiency of intracellular P could be furtherly improved by that the 0.75 mol/L LiCl solution was used to resuspend the bacterial cell before ultrasonic extraction (i.e., LiCl-ultrasonic protocol). The ultrasonic protocol was more suitable to extract the intracellular P and its poly-P of EBPR sludge than the other 4 protocols (i.e., PCA-NaOH, EDTA-NaOH, freezing-grind and LiCl-ultrasonic), which had the technical characteristics of (i) with relatively high extraction efficiency of intracellular P, (ii) without hydrolysis of intracellular poly-P, (iii) with weak noise signal in 31P NMR spectrum and (iv) with simple extraction process and short extraction time. It was founded by the ultrasonic protocol that there was the high content (82.88%–89.79% of intracellular P content) of intracellular poly-P with long average chain length (376.4–383.2) in the EBPR sludges. Importantly, it was confirmed that the EBPR process was related to the combined action of extracellular and intracellular poly-P using a new fractionation method of P in EBPR sludge, which included the ultrasonic protocol at high power density for extracting the intracellular P and its poly-P.